The goal of this proposal is to determine the molecular mechanisms by which tumor necrosis factor-alpha (TNF) and other cytokines induce cardiac dysfunction during burn shock. Previous work has demonstrated that TNF is secreted by cardiac cells following burn injury, and that inhibition of TNF following burn injury significantly ameliorates myocardial depression. The experiments proposed here will elucidate the molecular mechanisms responsible for myocardial depression and thereby provide a biologic basis for future clinical therapies. We propose that burn-induced cytokines act directly on myocytes to alter gene expression for proteins involved in calcium transport and other functions vital to normal contractility and relaxation. This hvpothesis will be tested by determining the effects of cytokines and other burn- induced products on the expression of genes encoding sarcoplasmic reticulum calcium ATPase (SERCA), the calcium efflux channel (ryanodine receptor), and phospholamban. In addition, the entire array of changes in gene expression will be systematically characterized by PCR-based differential display of myocyte cDNA before and after exposure to burn- induced mediators. Thus far, experiments have indicated that exposure of myocytes to cytokines induces expression of inducible nitric oxide synthase (iNOS). Through the use of promoter-reporter constructs, we will characterize the intracellular signaling and transcriptional activation required for iNOS induction in myocytes. The induction and expression of iNOS in cardiac cells following burn injury will then be examined by Northern and PCR analysis, as well as by assay of myocyte nitrite secretion in vitro. The secretagogue for TNF in the heart following burns is also unknown. To determine whether endotoxin is the stimulus for TNF secretion during burn shock, we will measure endotoxin in the portal and systemic venous plasma of burned guinea pigs; we will then neutralize endotoxin by continuous infusion of bacteriacidal increasing protein (BPI) following burn injury in vivo, and determine whether cardiac dysfunction is improved. Finally, to create a model in which to study the the effects of TNF and secondary mediators on the heart in vivo, and to establish a model in which therapies against cytokines can be tested, we will drive expression of the mouse TNF cDNA by a myoglobin promoter in transgenic mice. This model will be characterized molecularly, physiologically, and anatomically. At the conclusion of these studies, we will better understand the molecular pathogenesis of burn shock, and gain insights into the development of novel treatments for this condition.